Project Summary /Abstract Neonates are highly susceptible to infection and respond poorly to vaccination for reasons that are not well understood. Based on our published data, we believe that neonates are particularly vulnerable to repeat infections because their nave CD8+ T cells are intrinsically defective at differentiating into memory CD8+ T cells. A major goal of this grant is to identify the key gene regulatory networks that underlie cell-intrinsic differences between neonatal and adult CD8+ T cells. Since microRNAs (miRNAs) are developmentally regulated and required for CD8+ T cell function, we hypothesized that defective CD8+ T cell memory formation in early life may be due to differences in miRNA expression patterns between neonatal and adult CD8+ T cells. To test our hypothesis, we used next generation sequencing to identify mouse miRNAs that are differentially regulated in neonatal and adult CD8+ T cells throughout the response to infection. Surprisingly, our results indicated that differences in miRNA expression profiles were most pronounced prior to immunological challenge, suggesting that developmentally-regulated miRNAs do not operate by altering the fate of effector cells at the peak of the response. Instead, these miRNAs appear to set the activation threshold prior to infection, causing neonatal CD8+ T cells to differentiate more rapidly into effector cells and biasing them away from a memory precursor fate. We are particularly interested in two miRNAs (miR-29 and miR-130), which we believe play a major role in cell-intrinsic differences that exist between neonatal and adult CD8+ T cells in mice and humans. MiR-130 is preferentially expressed in neonatal CD8+ T cells and targets a number of genes involved in negative regulation of T cell proliferation or apoptosis. MiR-29, on the other hand, is more abundant in adult CD8+ T cells and regulates the expression of transcription factors involved in effector and memory cell differentiation. We propose that the miR-29/miR-130 axis acts as a developmental rheostat for adjusting the activation threshold of CD8+ T cells, controlling the balance between rapid effector cells (neonates) and long-lived memory cells (adults). The main objectives of this proposal are to determine how age-related changes in miR-29 and miR-130 expression alter the ability of CD8+ T cells to respond to infection (Aim 1); identify the key target genes regulated by miR- 29 and miR-130 prior to activation (Aim 2); and determine whether miR-29 and miR-130 can predict vaccine- specific CD8+ T cell responses in newborns (Aim 3). Accomplishing these aims will lend support for a new model describing how miRNAs regulate the CD8+ T cell response to infection, which can lead to novel therapeutic strategies for enhancing the development of memory CD8+ T cells in early life.